Chemical and Biological Investigation of Ferula tingitana L. Growing in Libya A Thesis submitted by Waleed Abourawi Alghwaji For the Degree of Master in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision Prof. Dr. Aly M. El Sayed Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University Prof. Dr. Kadriya Shafik El-Deeb Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University Associate. Prof. Dr. Abeer M. El- Sayed Associate Professor of Pharmacognosy Faculty of Pharmacy, Cairo University Pharmacognosy Department Faculty of Pharmacy Cairo University 2018 1 Abstract To contribute new knowledge to the currently existing known chemical/or biological data about Ferula tingitana L. growing in Libya. GC/MS of the lipoidal profile was investigated. Daucol is the major recognized sesquiterpene. Linolenic acid is the main makeup of fatty acids. The chemical composition of the essential oil of flower (0.06% w/v) and leaves (0.1% w/v) of F. tingitana was comparatively analyzed by GC-MS. Furthermore, to throw light on antimicrobial and cytotoxic activities of oils derived Libyan plant. The main constituents of both flower-and leaves- derived oil samples were α-thujene (13.5-2.3%), elemol (8.9- 8.3%), eudesmol (0.6-9.7%) and cadinol (2.2-13.8%) respectively. The principle difference was a considerably more pronounced sesquiterpenes presence in the leaves-oil, amounting to 74.0%, than in the flower counterpart (39.9%). Chromatographic analysis of the soluble fractions of the ethanolic extract were afforded β- sitosterol 1, colladonin 2, scopoletin 3, caffeic acid 4, sesquiterpene coumarin 5 and 1-(3,4-dihydroxycinnamoyl)cyclopentane-2`,3`- diol 6. Leaves-oil sample being mostly efficient as antibacterial against Bacillus subtilis and Neisseria gonorrhea with potency 48.3, 41.9% compared to tetracycline standard antibacterial drug. The essential oil samples revealed marked in vitro cytotoxicity against breast (MCF-7), cervical (HELA) and liver (HEPG2) carcinoma cell lines with IC50% (6.9, 4.8), (8.6, 10.9) and (4.4, 2 4.2) for the flower-,leaves- derived oil sample, respectively. In vitro cytotoxicity screening of the ethanol extract f F.tingitana on hormone responsive breast (MCF7), cervical (HELA) and liver carcinoma (HEPG2) cell lines was investigated. Ethanolic extract (50µg/ml) is prevailed a reduction in the levels of inflammatory mediators; NO, TNF-α, COX-2 and 5-LOX in Raw murine macrophage (RAW 264.7). As well as an alteration in the female reproductive hormones of adult female Wistar rats upon given 200, 300 mg/g b.wt of F. tingitana was evidenced. This is indicative of adverse effect on the menstruation, ovulation of follicles and consequently may impair fertility. Furthermore, a significant decline in biochemical serum level of glucose, triglyceride and cholesterol were observed. Keywords: Antimicrobial, apiaceae, antiinflammatory, cadinol, cytotoxicity, contraceptive, elemol, hypoglycemic, Ferula tingitana L., sesquiterpene coumarins. 3 Introduction Global use of herbs as therapeutic tool for treatment of human diseases is known since the early days of human kind. They continue to be important source in traditional medicine and worldwide for health promotion, According to WHO report approximately more than 25% of drugs distributed worldwide are derived from plants. They continue to be used in care problems because they are affordable and contain natural constituents, which are inherently safe effective and with minimum side effects .They can be prescribed in the form of crude extracts, which contain several active components with potentiating activity, which cannot be achieved, by any single components. Consequently, many plants and herbs are now day subjected to screening tests or research seeking natural products with interesting biological activity. Extracts from different species of the genus ferula (Apiaceae) have had various biological applications for decades. The promising biological features of these extracts such as cytotoxic, antibacterial, antiviral, p-glycoprotien (p-gp) inhibitor effect anti- inflammatory, have been attributed to the presence of sesquiterpene coumarins constituents, more over they have been utilized as a chemosystematics' markers such as those evidenced in population of F.communis where two chemo types have been recognized in the region of Sardinia. One chemo type contained 4 prenylated 4-hydroxy coumarins, While the other contained sesquiterpenes coumarin ethers. Chemical variants of different types of plants from different regions are the major target of study of many research groups. Species of ferula are mostly grown in mountain regions in different countries such as Russia, Pakistan, India and western Himalayas, Iran and China. However, none of these locations have reported the presence of F.tingitana L. among their species. Some ferula species are used as spice and many are known to be rich source of gum- resin which is prescribed in folklore medicine as anthelmentic, antiseptic, antispasmodic, analgesic, carminative, diuretic, expectorant and laxative properties. Members of ferula are herbaceous plants growing in Misilata Libya and have strong aromatic smell. Aim of work The aim of this work is to explore bio-active compounds from F.tingitana L. since the scientific validation of traditionally used plant in treatment is highly demanded. To contribute new knowledge to the currently existing known chemical or biological data about F.tingitana L. growing in Libya. 5 Review The unsaponifiable matter (USM) prepared from the saponifiable fraction were analyzed by GC/FID. The USM composition was dominated by hydrocarbons (6.6%) with octadecanal as major (1.6%); phytosterols were minor (35.5%) with stigmasterol (1.53%) as main component. and Sesquiterpene constituents were up to (24.22 %), Daucol was the major identified one (16.79%). The Identification of the fatty acids was accomplished by comparison of the retention times with standard fatty acid methyl esters. Quantitative determination was based on peak area measurement. The percentage of identified unsaturated fatty acids (FA) was doubled that of the saturated ones (51.4 vs 22.2%); the major was Linoleic acid unsaturated (36.7 %) followed by the saturated Palmitic acid (8.5%). Hydro-distillation of the flower and leaves of F.tingitana L. yielded 0.06%w/v and 0.10%w/v, respectively of clear colored oil exhibiting a characteristic pleasing odor. The yield of leaves derived oil exceed that obtained from flower about 2 times as flower-oil sample as depicted in Table 10 Within total 32 constituents could be recognized in the flower and 28 in leaves, amounted to 97.2% and 97.9% of the oil derived from the individual organs. To our knowledge, this is the first report 6 dealing with the chemical profile of volatile oil of Libyan F.tingitana L. flower, as confront to the leaf derived volatiles. To the best of our knowledge, this is the first report on the chemical composition of the essential oil of flower and leaves of F.tingitana L. growing in Libya. Nevertheless, the composition of the essential oil from F. tingitana L., cultivated in different regions of Turkey, was previously studied (Essential oil of Turkish F.tingitana L. was rich in monoterpenes represented by Naphthalene (15%), α-Pinene (11%). Meanwhile sesquiterpenes were predominated by Daucene (6%) (Z)-β-Farnesene (5%) Germacrene D (5%). Elemol, cadinol, α-thujene and α-terpinoline, the major components of the oil of flower and leaves in the present work and respectively were not detected in the Turkish sample (4). On the contrary, naphthalene (absent in the Libyan sample), Daucene (6%) (Z)-β-Farnesene (5%) Germacrene D (5%).were present in major quantities in the essential oil of the flower of F.tingitana L. of Turkey (4). Moreover, the hydrocarbons, β-caryophyllene, elemene, α- humulene, and eudesma-3,5diene were present Libyan samples. α- pinene was present in both Turkish and Libyan samples in comparable amounts. The differences may be due to climatic and other extrinsic conditions such as where and when the samples were collected. 7 The solvent-free petroleum ether extract of the air-dried aerial parts (15g) was subjected to vacuum liquid chromatography column (VLC) followed by repeated conventional column chromatography on silica gel; gradient elution was applied. Two compounds F1 and F2 were isolated and identified through determination of their physico-chemical, chromatographic and spectral data (IR, MS, and NMR), and via comparison with published ones. The structural formula of F1 was established as β-sitosterol and that of F2 as Colladonin.a sesquiterpine coumarin derivative. These two compounds are the reported before from the species. Summary The solvent-free methylene chloride extractives (15g) were fractionated on a series of chromatographic columns starting with VLC on silica gel followed by repeated chromatography on Sephadex LH-20column. This process resulted in isolation of three pure compounds F3, F4, and F5, which were identified through physico-chemical, chromatographic and spectral (NMR)data and as compared to those published compounds identified as Scopoletin, caffeic acid and sesquiterpene coumarin derivative, identified by (NMR HSQS and HMBC) respectively. 8 The dry ethyl acetate extract (4g) was subjected to chromatographed on Diaion column (3.5x50 cm), packed with Diaion (50g) followed by fractionation on Sephadex LH-20 columns to afford one pure compound F6. These were further identified via physico-chemical, chromatographic and